Flow cytometers are instruments that are commonly employed in the medical industry to analyze particles (e.g., blood cells) in a patient's body fluid as an adjunct to the diagnosis and treatment of disease. As a non-limiting example, during chemotherapy treatment, such instruments may be used to sort and collect healthy blood cells (stem cells) from a quantity of blood that has been removed from a patient's bone marrow prior to chemotherapy. Once a chemotherapy treatment session is completed, a collected quantity of these cells is the reinjected into the patient, to facilitate migration and healthy blood cell reproduction.
In accordance with the customary operation of a flow cytometer, particles to be analyzed, such as blood cells of a centrifuged blood sample are injected from a storage container into a (pressurized) continuous or uninterrupted stream of carrier fluid (e.g., saline) that travels through a carrier fluid transport chamber in which individual particles are sensed and contained within droplets that break off from the fluid stream exiting the fluid transport chamber. As diagrammatically illustrated in FIG. 1, which shows a portion of a fluid transport chamber 10 of a ‘SENSE IN QUARTZ’ flow cytometer system, a particle-containing carrier fluid 11 and its surrounding sheath fluid layer 12 are directed along an axial flow direction 13 from a relatively wide diameter portion 14 to a reduced diameter exit orifice 15 of the fluid flow chamber 10.
The particle-carrying fluid 11, that has been introduced into an upstream zone 21 of the chamber, is intersected at a particle-sensing zone 22 by an output (laser) beam 31 emitted by an optical illumination subsystem, such as one or more lasers 30. Located optically in the path of the laser output beam 31 after its being intercepted by the carrier fluid stream are one or more sensors of a photodetector subsystem 32. The photodetecting subsystem is positioned to receive light modulated by the contents of (particles/cells within) the carrier fluid stream, which typically includes light reflected off a cell, the blocking of light by a cell, and a light emission from a fluorescent dye antibody attached to a cell.
Downstream of the particle sensing zone 22 is a fluid stream constriction zone 23, wherein the cross sections of the carrier fluid stream and its surrounding sheath are reduced or constricted, so that the carrier fluid exits the chamber through an exit aperture or orifice 15 at a relatively high velocity relative to its travel within the chamber and enter an air space exit zone 24. From this location the constricted fluid stream, whose cross section is considerably smaller than during its travel through the fluid transport chamber and is sized to accommodate a single particle, continues on through a droplet separation and charging zone 25, where a charge is selectively applied to a droplet as the droplet separates or breaks off the fluid stream 11 proper.
Conventional SENSE IN QUARTZ technology systems of the type shown in FIG. 1 incorporate a fixed time delay period between the time a particle is sensed and analyzed in the sensing zone 22 and the time of the application of a droplet sorting charge in the downstream charging and droplet separation zone 25. The use of a fixed delay constitutes a source of error in the charging/sorting operation due to the fact that not all particles travel at the same effective speed along the transport direction of the fluid, due to the fact that not all particles traverse the same trajectory through the flow chamber.
More particularly, established flow inside a chamber can be generally described as parabolic flow to some degree. In parabolic flow within zone 21 there is a relationship between particles flowing along the central axis relative to those particles traveling closer to a wall of the chamber. These particles will continue at their respective (parabola profile based) velocities unless acted upon by an outside influence. Such an influence is created when the fluid flow is forced through a change in geometry, such as the exit orifice 15. Just upstream of this orifice, there is a constriction of the flow and a subsequent acceleration of the particles relative to the change in cross-sectional area. This acceleration is not uniform and therefore causes a greater acceleration of the particles depending upon wherein the particles are flowing in the fluid stream, thereby separating particles at a faster rate than that occurring in the sensing zone 22 of the chamber. Once the flow exits the area affected by the exit orifice, at zone 24, the velocity can be assumed to be constant, so that there is no further separation of the particles.
This differential in velocity and trajectories may be readily understood by referring to FIG. 2, which shows a first particle A traveling along the axis 13 of the carrier fluid channel, and a second particle B that is displaced by some distance from the axis 13 as it travels through the fluid transport chamber. Superimposed on the fluid transport chamber diagram of FIG. 2 is a set of velocity profiles showing three examples of different velocities associated with different positions of particles relative to the fluid transport axis 13. In the illustrated example, within the zone 21, the particle A has a velocity V1A which is represented by a velocity vector (arrow 41), that coincides with the peak value of a generally parabolic velocity profile of the speed of travel of the carrier fluid through the upstream portion of chamber.
Similarly, within the zone 21, the (off-axis) particle B has a velocity V1B, which is represented by a reduced amplitude velocity vector (arrow 42), which coincides with a reduced value along the velocity profile of the speed of travel of the carrier fluid through the chamber. FIG. 2 also shows within the zone 21 a further (off-axis) velocity V1C that lies in between associated the velocities V1A and V1B, and would be associated with an off axis particle (not shown) lying between the coaxial particle A and the off-axis particle B.
From FIG. 2 it is apparent that particles closer to the axis 13 travel at higher velocities as they pass through the sensing zone 22. The velocity profile overlay of FIG. 2 also shows that as the particles leave the sensing zone 22 and approach an acceleration zone 23 adjacent to the exit orifice 15, the speed of the fluid containing the particles must increase in order to comply with the conservation of mass. For the particle A, its velocity increases from a value of V1A in the sensing zone 22 along a first acceleration profile f1A to the entrance zone 23 to the exit orifice 15. Thereafter, the velocity of particle A increases (accelerates) slightly along a second acceleration profile f2A(a) as its passes through the orifice 15 and travels through the freespace region from zone 24 immediately adjacent to the downstream side of orifice 15 to a downstream zone 25 at a final velocity of V2A. Likewise, the velocity of particle B velocity increases from a value of V1B at the sensing zone along a first acceleration profile f1B(a), reaching a higher velocity at the entrance zone 23 to the exit orifice 15. Thereafter, the velocity of particle B increases (accelerates) slightly along a second acceleration profile f2B(a) as its passes through the orifice 15 and travels through the freespace region from zone 24 immediately adjacent to the downstream side of orifice 15 to a downstream zone 25 reaching a final velocity of V2B.
From these different velocity profiles it can be seen that the different trajectories of particles A and B cause different arrival times at the point of application of a droplet sorting charge in zone 25, which is downstream of exit orifice 15, so that a fixed time delay will not guarantee that the correct particle will be contained in the sorted droplet. In order to maintain conservation of mass, an off-axis particle, such as the particle B, must undergo a more rapid acceleration than the acceleration of particle A, as it approaches zone 23, so that it will exit the chamber 10 at the speed of the fluid stream. If the two particles A and B are in the sensing zone 22 at the same time, the coaxial particle A, which has the higher velocity, will exit the chamber ahead of the slower particle B, which has to accelerate up to the speed of the fluid stream exiting the chamber at orifice 15. In an attempt to deal with this problem, conventional systems sort more than one droplet; this, in turn, decreases the purity of the sorted populations and increases the dilution of the particles.